Method for regulating tomato wound bud de novo regeneration by slnac2 gene and application

Abstract

The application provides a method and application of SlNAC2 gene in regulating tomato wound bud de novo regeneration, and belongs to the technical field of plant genetic engineering. The complete coding sequence of SlNAC2 gene is obtained from a tomato genome, a specific sgRNA target point is designed, and the target point is constructed on a CRISPR / Cas9 gene editing carrier. A recombinant carrier is introduced into tomato cotyledon explants by using an agrobacterium-mediated genetic transformation method, and finally, a mutant plant with a homozygous knockout of the SlNAC2 gene is obtained. The application first discloses the positive regulation of the SlNAC2 gene in regulating tomato hypocotyl bud regeneration, and establishes a high-efficiency and stable genetic improvement method, which provides important gene resources and breeding strategies for creating gene editing breeding independent of genotypes.

Description

Technical Field

[0001] This invention belongs to the field of plant genetic engineering technology, specifically relating to a gene that promotes de novo regeneration of tomato organs and its regulation method, particularly the method and application of SlNAC2 gene regulating de novo regeneration of tomato wound buds. Background Technology

[0002] Plant regeneration refers to the process by which plant tissues or organs maintain their functional integrity through self-repair or structural replacement after being damaged or subjected to environmental stress. De novo organ regeneration is a crucial strategy for plants to cope with damage and maintain individual survival, and it is also a core foundation for crop genetic improvement. Under in vitro culture conditions, plant cells, tissues, or organs can be induced to form complete plants, achieving a systematic reconstruction from local to overall. From traditional agricultural methods like cuttings and grafting to modern biotechnology techniques such as tissue culture and somatic embryogenesis, all rely on the regenerative capacity of plants.

[0003] Tomato (Solanum lycopersicum) is one of the world's three major traded vegetables, with China accounting for approximately one-third of global tomato production. Currently, the prevalent genotype dependence in tomato genetic transformation has become a significant technical bottleneck restricting its bio-breeding efficiency. Therefore, screening for key factors that effectively promote regeneration and breaking down genotype dependence barriers is crucial for improving the regeneration efficiency of tomatoes with different genotypes and expanding the application of precision breeding technologies such as genetic transformation and gene editing (e.g., CRISPR). This will help accelerate the cultivation of high-yielding, high-quality, and disease-resistant tomato varieties, providing technical support for achieving self-sufficiency in seed sources and ensuring national food security. Furthermore, related regeneration-promoting factors are also expected to be extended to other plant species with generally low genetic transformation efficiency. Summary of the Invention

[0004] The purpose of this invention is to provide a method and application for regulating the de novo regeneration of tomato wound buds using the SlNAC2 gene. By using CRISPR / Cas9 gene editing technology to target and knock out the endogenous tomato gene SlNAC2, the regeneration of tomato hypocotyl wound buds is significantly inhibited, providing a new technical approach for improving tomato quality and showing important application prospects in the field of bio-breeding.

[0005] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0006] This invention provides a method for regulating de novo regeneration of tomato wound buds using the SlNAC2 gene, comprising the following steps:

[0007] Step 1: Two sgRNA targets, sgRNA1 and sgRNA2, were screened from the SlNAC2 gene. The nucleotide sequence of sgRNA1 is shown in SEQ ID NO.3, and the nucleotide sequence of sgRNA2 is shown in SEQ ID NO.4.

[0008] Step 2: Design specific primers based on the sgRNA target and perform primer annealing.

[0009] Step 3: Ligate the product from Step 2 with the enzyme-digested vector to construct the CRISPR / Cas9-SlNAC2 recombinant vector;

[0010] Step 4: The CRISPR / Cas9-SlNAC2 recombinant vector constructed in Step 3 is transferred into Agrobacterium tumefaciens and then into the target tomato cotyledon explants via Agrobacterium infection.

[0011] Furthermore, in step two, the sequence of the specific primer is as follows:

[0012] SlNAC2-sgRNA1-F: 5′-TGATTGCAGCAATTGGAGTTACCGG-3′ (SEQ ID NO.5);

[0013] SlNAC2-sgRNA1-R: 5′-AAACCCGGTAACTCCAATTGCTGCA-3′ (SEQ ID NO. 6);

[0014] SlNAC2-sgRNA2-F: 5′-TGATTGGGTGCAGCACTATCTCTGC-3′ (SEQ ID NO.7);

[0015] SlNAC2-sgRNA2-R: 5′-AAGCAGAGATAGTGCTGCACCCA-3′ (SEQ ID NO. 8).

[0016] Furthermore, in step two, the forward and reverse primers are incubated at 95°C for 5 min in a PCR instrument, annealed to 25°C at a temperature of 0.1°C / s, and then cooled to room temperature to complete the annealing.

[0017] Furthermore, in step three, the digested vector is a p1300-AtU6-35S-Cas9 vector digested with BsaI enzyme.

[0018] Furthermore, in step four, the target tomato cotyledon explant is a tomato cotyledon with two cotyledons that has been sown for two weeks.

[0019] Furthermore, step four includes:

[0020] 1) Agrobacterium infection: Tomato cotyledons with 2 cotyledons that were pre-cultured 2 weeks after sowing were cut into 1 square centimeter pieces and infected in MS infection solution for 15 min. After being dried with sterile filter paper, they were placed in new pre-culture medium and co-cultured in the dark at 25°C for 48 h.

[0021] 2) Differentiation culture: Transfer the explants to the differentiation medium and culture at 25°C with 16h light / 8h dark, changing the medium every 15 days;

[0022] 3) Rooting culture: Cut off differentiated buds and insert them into the rooting culture medium. After rooting, transplant them into nutrient pots.

[0023] Furthermore, the MS infection solution was formulated as follows: MS powder 4.3 g / L, sucrose 20 g / L, pH 6.

[0024] Furthermore, the pre-medium formulation was as follows: MS powder 4.3 g / L, sucrose 30 g / L, agar 8 g / L, 2,4-D (2,4-dichlorophenoxyacetic acid) 0.2 mg / L, pH 5.5; the differentiation medium formulation was as follows: MS powder 4.3 g / L, sucrose 20 g / L, agar 7.4 g / L, zeatin 2 mg / L, termetin 300 mg / L, hygromycin 6 mg / L, pH 6; and the rooting medium formulation was as follows: MS powder 4.3 g / L, sucrose 20 g / L, agar 7.4 g / L, auxin 5 mg / L, termetin 300 mg / L, hygromycin 6 mg / L, pH 6.

[0025] The present invention also provides an application of the SlNAC2 gene in regulating de novo regeneration of tomato wound buds, the nucleotide sequence of which is shown in SEQ ID NO.1.

[0026] Furthermore, the application includes: significantly inhibiting bud regeneration after treatment of hypocotyl wounds in tomato by knocking out the tomato SlNAC2 gene.

[0027] The beneficial effects of this invention are as follows:

[0028] This invention obtained the complete coding sequence of the SlNAC2 gene from the tomato genome, designed a specific sgRNA target, and constructed it into a CRISPR / Cas9 gene editing vector. Using Agrobacterium-mediated genetic transformation, the recombinant vector was introduced into tomato cotyledon explants, ultimately obtaining homozygous knockout mutant plants of the SlNAC2 gene. Analysis of the fruits of the mutant plants showed that, compared with wild-type tomatoes, the SlNAC2 gene knockout mutant significantly inhibited hypocotyl bud regeneration. This invention reveals for the first time the positive regulatory role of the SlNAC2 gene in regulating hypocotyl bud regeneration in tomatoes and establishes an efficient and stable genetic improvement method, providing important genetic resources and breeding strategies for creating non-genotype-dependent gene editing (such as CRISPR) breeding. Attached Figure Description

[0029] Figure 1 The PCR sequencing results of leaves from wild tomatoes and SlNAC2 gene knockout tomato lines showed that slnac2-cr1 and slnac2-cr2 are two SlNAC2 gene knockout lines.

[0030] Figure 2 Comparison of hypocotyl shoot regeneration between wild tomato and SlNAC2 gene knockout tomato. WT is a wild-type tomato, while slnac2-cr1 and slnac2-cr2 are two SlNAC2 gene knockout lines. Detailed Implementation

[0031] To more clearly illustrate the present invention, the invention will be further described in detail below with reference to embodiments and accompanying drawings. Those skilled in the art should understand that the specific descriptions and figures below are illustrative and not intended to limit the scope of the invention, nor should they be used to restrict the scope of protection of the invention.

[0032] Example

[0033] I. Obtaining strain EHA105 containing the CRISPR / Cas9-SlNAC2 recombinant plasmid

[0034] Step 1: Target sequence design for the SlNAC2 gene

[0035] The mature sequence of the tomato SlNAC2 gene was obtained from the tomato genome database of the phytozome website. Its CDS sequence is shown in SEQ ID NO.1, and its amino acid sequence is shown in SEQ ID NO.2. The tomato SlNAC2 gene is 906 bp in length and consists of three exons. The sgRNA of the SlNAC2 gene was screened using the online CRISPR-P tool (http: / / cbi.hzau.edu.cn / cgi-bin / CRISPR). The sgRNA target sites designed in this application are located on the first exon of the gene's CDS, with a GC content in the range of 45%-70%, and differing from other locations in the genome by at least three bases. The two designed target sequences are sgRNA1: 5′-CAGCAATTGGAGTTACCGG-3′ (SEQ ID NO.3) and sgRNA2: 5′-GGTGCAGCACTATCTCTGC-3′ (SEQ ID NO.4).

[0036] Step 2: Primer synthesis

[0037] The following primer sequences were synthesized by Shanghai Bioengineering Co., Ltd.:

[0038] SlNAC2-sgRNA1-F:5′- TGATTG CAGCAATTGGAGTTACCGG-3′ (SEQ ID NO.5);

[0039] SlNAC2-sgRNA1-R: 5′- AAAC CCGGTAACTCCAATTGCTG CA -3′(SEQ ID NO.6)

[0040] SlNAC2-sgRNA2-F:5′- TGATTG GGTGCAGCACTATCTCTGC-3′ (SEQ ID NO.7);

[0041] SlNAC2-sgRNA2-R:5′- AA GCAGAGATAGTGCTGCACC CA -3′ (SEQ ID NO.8);

[0042] The underlined part represents the connector sequence.

[0043] Step 3: Primer annealing

[0044] 10 μL each of the 10 μmol / L forward and reverse primers from step 2 were incubated at 95°C for 5 min in a PCR instrument, annealed to 25°C at a temperature of 0.1°C / s, and then 180 μL of ddH2O was added. The annealing was then completed by cooling to room temperature.

[0045] Step 4: Vector digestion

[0046] The p1300-AtU6-35S-Cas9 vector was digested with BsaⅠ enzyme (NEB) at 37℃ for 4-6 h. The reaction system was as follows: 1 μg p1300-AtU6-35S-Cas9 vector, 5 μL 10× BasⅠ CutSmart buffer, 1 μL BsaⅠ enzyme (NEB), and ddH2O to a final volume of 50 μL.

[0047] Step 5: Ligation reaction

[0048] The product from step 3 was ligated into the digested p1300-AtU6-35S-Cas9 vector using T4 ligase. The reaction mixture consisted of 5 μL of the product from step 3, 1 μL of the product from step 4, 1 μL of 10×T4 ligase buffer, and 0.5 μL of T4 ligase (thermo). The reaction was carried out at 16°C for 2 h.

[0049] Step 6: E. coli transformation

[0050] Add 1 μL of the ligation product to 25 μL of DH5α competent cells, incubate on ice for 25 min, then heat shock at 42°C for 45 s, and immediately incubate on ice for 2 min. Add 900 μL of antibiotic-free LB medium and incubate at 37°C with shaking for 1 h. Centrifuge at 13000 rpm / s for 1 min, discard the supernatant, and aspirate the remaining 50 μL of bacterial culture, spread it evenly on LB agar plates containing kanamycin-resistant culture, and incubate at 37°C for 16 h.

[0051] Step 7: Identification of positive monoclonal colonies

[0052] Single colonies were selected for PCR verification. The upstream primer was the universal M13 primer. The downstream primers were SlNAC2-sgRNA1-R′: 5′-CCGGTAACTCCAATTGCTG-3′ (SEQ ID NO.9) and SlNAC2-sgRNA2-R′: 5′-GCAGAGATAGTGCTGCACC-3′ (SEQ ID NO.10).

[0053] After verification by agarose gel electrophoresis, the positive clone plasmid was extracted and sent for sequencing (Shanghai Bioengineering Co., Ltd.), and the CRISPR / Cas9-SlNAC2 recombinant plasmid was obtained.

[0054] Step 8: Transform the CRISPR / Cas9-SlNAC2 recombinant plasmid into EHA105 Agrobacterium competent cells to obtain EHA105 strain containing the CRISPR / Cas9-SlNAC2 plasmid.

[0055] II. Construction of SlNAC2 gene mutant plants

[0056] The CRISPR / Cas9-SlNAC2 vector was transformed into tomato cotyledons using the leaf disc method.

[0057] The specific steps are as follows:

[0058] 1) Agrobacterium infection: Tomato cotyledons with 2 cotyledons that were pre-cultured 2 weeks after sowing were cut into 1 square centimeter pieces and soaked in 15 mL MS infection solution for 15 min. After being dried with sterile filter paper, they were placed in a new pre-culture medium and co-cultured in the dark at 25°C for 48 h.

[0059] 2) Differentiation culture: Transfer the explants to the differentiation medium and culture at 25°C with 16h light / 8h darkness. Change the medium every 15 days.

[0060] 3) Rooting culture: Cut off differentiated buds and insert them into rooting culture medium. After rooting, transplant them into nutrient pots.

[0061] Specific methods for preparing culture media:

[0062] Seed germination medium: MS powder (PhytoTech) 2.15 g / L, sucrose 10 g / L, agar 7.4 g / L, pH 5.8.

[0063] Pre-medium: MS powder 4.3 g / L, sucrose 30 g / L, agar 8 g / L, 2,4-D (2,4-dichlorophenoxyacetic acid) 0.2 mg / L, pH 5.5.

[0064] Differentiation medium: MS powder 4.3 g / L, sucrose 20 g / L, agar 7.4 g / L, zeatin 2 mg / L, termethin 300 mg / L, hygromycin 6 mg / L, pH 6.

[0065] Rooting medium: MS powder 4.3 g / L, sucrose 20 g / L, agar 7.4 g / L, auxin 5 mg / L, termethin 300 mg / L, hygromycin 6 mg / L, pH 6.

[0066] MS infection solution: MS powder 4.3 g / L, sucrose 20 g / L, pH 6.

[0067] LB liquid medium: NaCl 10 g / L, tryptone 10 g / L, yeast extract 5 g / L, pH 7.

[0068] III. Molecular Identification of SlNAC2 Mutants

[0069] 1) DNA extraction and PCR analysis

[0070] DNA was extracted from leaves of T0 generation plants using the CTAB method. Specific primers were designed upstream and downstream of the sgRNA sequence location of the SlNAC2 gene.

[0071] SlNAC2-CHECK-F: 5′-AGGAGCAGCAACAAACAGAGA-3′ (SEQ ID NO. 11);

[0072] SlNAC2-CHECK-R: 5′-GAACAGATCCAAAAACTGTGATCA-3′ (SEQ ID NO. 12).

[0073] Mutant screening was conducted. Seeds from the heterozygous T0 generation, based on PCR sequencing results, were sown again to analyze the editing status of the T1 generation plants. PCR sequencing alignment results showed that slnac2-cr1 had a 53 bp deletion in its first exon, while slnac2-cr2 had a 4 bp deletion and a 1 bp insertion in its first exon. (See...) Figure 1 .

[0074] 2) Determination of hypocotyl bud regeneration capacity

[0075] Experiments were conducted using homozygous seeds from F2 and later generations to statistically analyze the differences in hypocotyl bud regeneration between wild-type and mutant tomato plants.

[0076] Two weeks after sowing F2 wild-type WT seeds and tomato SlNAC2 mutants (slnac2-cr1, slnac2-cr2) on a culture medium, tomato seedlings with two cotyledons were cut at approximately two-thirds of the distance from the cotyledons, removing the upper part, including the cotyledons and apical meristem. The remaining hypocotyls with roots were cultured. Observation after culture revealed that the wild-type plants had regenerated complete bud structures at the wound site after 12 days (12D), but the two mutant materials had not sprouted after 16 days (16D).

[0077] The results showed that knocking out the tomato SlNAC2 gene significantly inhibited shoot regeneration after treatment with wounds on the hypocotyl of the tomato cotyledons.

[0078] The embodiments of the present invention are for illustrative purposes only and are not intended to limit the implementation. Those skilled in the art can make various modifications and variations based on the present invention, and these variations still fall within the protection scope of the present invention.

Claims

1. A method for regulating de novo regeneration of tomato wound buds using the SlNAC2 gene, characterized by: Includes the following steps: Step 1: Two sgRNA targets, sgRNA1 and sgRNA2, were screened from the SlNAC2 gene. The nucleotide sequence of sgRNA1 is shown in SEQ ID NO.3, and the nucleotide sequence of sgRNA2 is shown in SEQ ID NO.

4. Step 2: Design specific primers based on the sgRNA target and perform primer annealing. Step 3: Ligate the product from Step 2 with the enzyme-digested vector to construct the CRISPR / Cas9-SlNAC2 recombinant vector; Step 4: The CRISPR / Cas9-SlNAC2 recombinant vector constructed in Step 3 is transferred into Agrobacterium tumefaciens and then into the target tomato cotyledon explants via Agrobacterium infection.

2. The method according to claim 1, characterized in that, In step two, the sequences of the specific primers are as follows: SlNAC2-sgRNA1-F: 5′-TGATTGCAGCAATTGGAGTTACCGG-3′ (SEQ ID NO.5); SlNAC2-sgRNA1-R: 5′-AAACCCGGTAACTCCAATTGCTGCA-3′ (SEQ ID NO. 6); SlNAC2-sgRNA2-F: 5′-TGATTGGGTGCAGCACTATCTCTGC-3′ (SEQ ID NO.7); SlNAC2-sgRNA2-R: 5′-AAGCAGAGATAGTGCTGCACCCA-3′ (SEQ ID NO. 8).

3. The method according to claim 1, characterized in that, In step two, the forward and reverse primers are incubated at 95°C for 5 min in a PCR instrument, annealed to 25°C at a temperature of 0.1°C / s, and then cooled to room temperature to complete the annealing.

4. The method according to claim 1, characterized in that, In step three, the digested vector is the p1300-AtU6-35S-Cas9 vector digested with BsaI enzyme.

5. The method according to claim 1, characterized in that, In step four, the target tomato cotyledon explant is a tomato cotyledon with two cotyledons that has been sown for two weeks.

6. The method according to claim 1, characterized in that, Step four includes: 1) Agrobacterium infection: Tomato cotyledons with 2 cotyledons that were pre-cultured 2 weeks after sowing were cut into 1 square centimeter pieces and infected in MS infection solution for 15 min. After being dried with sterile filter paper, they were placed in new pre-culture medium and co-cultured in the dark at 25°C for 48 h. 2) Differentiation culture: Transfer the explants to the differentiation medium and culture at 25°C with 16h light / 8h dark, changing the medium every 15 days; 3) Rooting culture: Cut off differentiated buds and insert them into the rooting culture medium. After rooting, transplant them into nutrient pots.

7. The method according to claim 6, characterized in that, The MS infection solution was formulated as follows: MS powder 4.3 g / L, sucrose 20 g / L, pH 6.

8. The method according to claim 6, characterized in that, The pre-medium formulation was as follows: MS powder 4.3 g / L, sucrose 30 g / L, agar 8 g / L, 2,4-D (2,4-dichlorophenoxyacetic acid) 0.2 mg / L, pH 5.5; the differentiation medium formulation was as follows: MS powder 4.3 g / L, sucrose 20 g / L, agar 7.4 g / L, zeatin 2 mg / L, termetin 300 mg / L, hygromycin 6 mg / L, pH 6; the rooting medium formulation was as follows: MS powder 4.3 g / L, sucrose 20 g / L, agar 7.4 g / L, auxin 5 mg / L, termetin 300 mg / L, hygromycin 6 mg / L, pH 6.

9. Application of SlNAC2 gene regulation of de novo regeneration of tomato wound buds, characterized by: The nucleotide sequence of the SlNAC2 gene is shown in SEQ ID NO.

1.

10. The application according to claim 9, characterized in that, The application includes: significantly inhibiting bud regeneration after treatment of hypocotyl wounds in tomato by knocking out the tomato SlNAC2 gene.

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